Each of the two promoters of the gal operon of E. coli is negatively regulated by two repressors: the classical Gal repressor and the newly discovered Gal isorepressor. Both act by binding to the same two spatially separated operators, OE an OI. Gal repressor and isorepressor belong to a family of bacterial regulatory proteins, we termed GalR family. An alignment of the proteins of the family show 60% homology throughout the entire sequences. We have established that both repressor and isorepressor participate in the modulation of a gal regulon, which includes, besides the gal operon, the mgl operon encoding one of the galactose transport systems and galR and galS, the genes for Gal repressor and isorepressor respectively. The isorepressor has a major and the repressor a minor role in the negative control of the mgl operon. The latter carries a gal operator element at the -60 region. The effect of isorepressor on mgl is at the level of transcription initiation. An in vitro transcription assay has been developed to study regulation with purified proteins. It uses supercoiled DNA minicircles which carry only the gal promoter segment. In this system, using DNA minicircle containing the gal promoters with lac operator sequences at OE and OI, we have demonstrated that requirement of DNA looping in vitro. Lac repressor, which is capable of association into a tetramer and forming a DNA loop as observed by electron microscopy, repressed transcription from P1 and P2 while a nonlooping Lac repressor mutant failed to show normal repression from both promoters. Repression mediated by DNA looping inhibited the synthesis of complete as well as abortive transcripts, demonstrating that the repression was on the formation or activity of the initial transcribing complex. DNase foot printing results indicated that repressor does not inhibit RNA polymerase binding.